The invention relates to the use of progesterone to reduce coronary artery reactivity, as well as kits relating thereto. The invention also involves screening methods and animal models for testing the ability of a compound to reduce coronary artery reactivity.
Cardiovascular disease, including coronary heart disease, stroke and other vascular diseases, is the leading cause of death of men and women in economically-developed countries. The most common and lethal form of cardiovascular disease is ischemic heart disease. It has generally been regarded that ischemic heart disease is caused, primarily, by atherosclerosis of the coronary arteries. This is a condition where plaques form in the inner lining of the arteries, causing narrowing of the channel and thereby impairing blood flow to the heart.
An increased risk for ischemic heart disease is observed in women after menopause or ovariectomy and presents a major medical challenge. It is the leading cause of death in postmenopausal women, which implicates the importance of the loss of ovarian steroid hormones.
Since it is understood that the formation of plaques is reduced, or even reversed by a high ratio of high density lipoproteins (HDL) to low density lipoproteins (LDL), it has been a strategy to inhibit ischemic heart disease by attempting to increase this ratio in the blood stream. Estrogen, an ovarian steroid hormone, has been observed to increase the HDL/LDL ratio, and studies so far suggest that estrogen replacement therapy for post-menopausal women decreases the incidence of coronary artery disease, myocardial infarction and related cardiovascular events by up to 50%.
The mechanism of estrogen""s cardioprotective effects is not completely established, although a favorable impact on the circulating lipid and lipoprotein profiles is postulated as central to estrogen""s cardioprotective effects. This explanation, however, is acknowledged to account for only a minority of estrogen""s potential cardioprotective effects.
One important issue is the danger of using unopposed estrogen, which is believed to increase the risk of developing endometrial bleeding, endometrial cancer, or breast cancer. Progestins, including specially synthetic progestins, have been found to oppose these undesirable effects of estrogen replacement. In particular, progestins have been shown to stop bleeding and reduce the risk of cancer in women receiving estrogen as hormone replacement therapy. But even this use of progestins is controversial, in that at least one study indicated that the combination of estrogen and progestin appeared to have a greater stimulatory effect on cell division than estrogen alone, suggesting that the risk of breast cancer is increased when estrogen is combined with progesterone. (See U.S. Pat. No 5,543,150). Likewise, it has been reported that progesterone and synthetic analogs""such as medroxyprogesterone acetate (MPA) cause modest reductions in serum HDL levels.
A variety of synthetics progestins were developed because the natural hormone progesterone (the chemically explicit and unique name of a defined steroid molecule) is quickly metabolized in the liver. Oral dosages of progesterone are typically very high and involve flooding the liver with progesterone, whereby a fraction of the dose escapes metabolism and enters the blood stream. Not only is an extraordinary high dose necessary (200 milligrams per day or more), there also is evidence that hepatic progesterone metabolites have adverse side effects.
Various attempts have been made at other delivery forms for progesterone, although such attempts have been largely unsuccessful because of undesirable fluctuation in serum levels, inconvenience of administration, and the like. Typically, when administered, progesterone is given in amounts to produce serum levels of at least 6 nanograms/ml, and typically to produce in the range of 10 nanograms/ml to 12 nanograms/ml.
It was indicated 20 years ago that progesterone can inhibit hypoxia-induced vasospasm, which resulted in the proposal that prostaglandin inhibitors such as indomethacin or chloroquine, nitrites and prostacyclins could be used to prevent cardiac damage from such vasospasm. It was not stated what levels of prtogesterone were administered to inhibit such hypoxia-induced vasospasm and progesterone itself was not suggested as a therapeutic to inhibit such vasospasm; perhaps because of the controversy surrounding the use of progesterone as a therapeutic agent. M. Karmazyn, et al., The Mechanism of Coronary Artery Spasm: Roles of Oxygen, Prostaglandins, Sex Hormones in Smoking, Medical Hypothesis 5:447-452 (1979).
A vasospasm is an abnormally strong and persistent contraction of the muscles of the coronary arteries which leads to transmural myocardial ischemia and can result in sudden cardiac death. The role of coronary vasospasm in cardiovascular disease is still controversial, and approaches to treatments for cardiovascular disease have not focused upon methods for reducing coronary vasospasm. Instead, it is generally believed that coronary vasospasm is caused by local injury to vessels, such as results from atherosclerosis and other structural injury, and that long-term treatment of cardiovascular disease requires prevention of atherosclerotic plaques, not treatments to prevent vasospasm.
U.S. Pat. No. 5,543,150 relates to a vaginal administration of progesterone to prevent endometrial cancer. The ""150 patent teaches that the local concentration of progesterone in the endometrial tissue is on the order of 7 nanograms/ml, which is sufficient to produce secretory transformation of the endometrium. The ""150 patent states that the systemic concentration of progesterone in the blood stream, remote from the endometrium, is between 1 and 2 nanograms/ml. According to the ""150 patent, the beneficial endometrial effects of progesterone are obtained at levels of 7 nanograms/ml, while the risk of breast cancer presented by progesterone therapy is remote because serum levels are only about 1 to 2 nanograms/ml, lower than those known to have any side-effects remote from the endometrium. It specifically is stated that the coronary vasodilating effect of estrogen is not reversed by such concentrations of progesterone, i.e., there was no indication of an adverse effect of progesterone.
A significant need exists for methods to determine the causes of coronary vasospasms, the factors contributing to the development of coronary vasospasms, and therapies that are effective in treating and in preventing coronary vasospasms. Consequently, the need exists for a reliable method to produce vasospasms, both in vivo in an animal model of the human disease and in vitro. This is necessary because coronary vasospasms are a transient pathologic occurrence, often with no observable gross or histopathologic lesions. Therefore, without the ability to reliably provoke a coronary vasospasm or its in vitro equivalent, it is difficult, if even possible, to reliably test potential preventative or therapeutic modalities for coronary vasospasms.
It has been discovered, surprisingly, that very low levels of progesterone can inhibit coronary artery reactivity and, therefore, can be used to inhibit certain adverse cardiovascular events and disorders. Also surprisingly, progesteronels protective effect was in contrast to the effects seen with the synthetic progestin MPA, in that MPA increased coronary artery reactivity. It also has been discovered that coronary artery reactivity is independent of vascular injury and can be mimicked in an animal free of plaques and known vascular injury. It further was discovered, surprisingly, that progesterone not only mediates a genomic effect on coronary arteries, but also exerts a direct effect on coronary arteries.
According to one aspect of the invention, a method is provided for reducing in a human subject coronary artery reactivity, to a vasoconstrictive substance. The method involves administering to the subject progesterone in an amount to achieve blood levels of progesterone of between 0.1 nanograms/ml and less than 4 nanograms/ml for at least 4 hours per day, and wherein said amount results in peak levels of progesterone of less than 6 nanograms/ml. In preferred embodiments, the blood levels of progesterone are between 1 nanogram/ml and less than 4 nanograms/ml, and most preferably between 1 nanogram/ml and 2 nanograms/ml. Even more preferably, the progesterone is administered in an amount to achieve such blood levels for at least 6 and preferably 12 or more hours per day.
The progesterone can be administered in a manner to achieve continuously such blood levels for periods of 1 day, 2 days, 3 days or more. It is contemplated that the treatment will be applied daily over a period of months without interruption. The most preferred dosage form is a topical preparation applied to the epidermis. Such a preparation avoids the first-pass through the liver, and the skin acts as a slow-release mechanism for providing the above-noted amounts of progesterone. Surprisingly, it has been determined that a single topical application, once each day, can result in the desired levels of progesterone for more than 4, 6, 12 and even up to 24 hours, without exceeding the peak levels (6 nanograms/ml). Another form of administration can be an implant containing sufficient amounts of progesterone to achieve the blood levels for at least 3 days, and preferably 30 days or more.
As mentioned above, the progesterone can inhibit coronary artery vasospasm in subjects that are free of observable plaques, who are apparently healthy and have no sign of vascular disease. Thus, the invention can be useful in subjects who are non-hypercholesterolemic. The subjects also may be nonarthritic and/or may be post-menopausal or ovariectomized.
According to another aspect of the invention, a method is provided for reducing in a female human subject, coronary artery reactivity to a vasoconstrictive substance. The method involves administering to the epidermis of the subject a topical preparation containing between 10 milligrams and 100 milligrams of progesterone. In some embodiments, the preparation contains between 20 and 50 milligrams of progesterone. The preparation can be administered only twice per day, preferably at 12 hours intervals. The preparation can even be administered only once per day and still achieve the desired therapeutic benefit.
According to another aspect of the invention, a kit is provided for dispensing a pharmaceutical. The kit includes a package which houses a container, a topical preparation containing progesterone in the container, a dispenser for dispensing a metered amount of the topical preparation, and instructions. The instructions are for dispensing an amount of the preparation which amount, when applied topically to the skin, achieves blood levels of progesterone of between 0.1 nanograms/ml and less than 4 nanograms/ml for at least 4 hours per day and, wherein said amount results in peak blood levels of progesterone of less than 6 nanograms/ml after said application. In one embodiment, the instructions are for dispensing between 10 and 100 milligrams of progesterone for daily application to the epidermis. In another embodiment, the instructions are for dispensing between 20 and 50 milligrams of progesterone for daily application to the epidermis. Preferably, the instructions are for dispensing an amount which achieves blood levels of between 1 nanogram/ml and 2 nanograms/ml for at least 6 hours per day. In one embodiment, the instructions are for daily application only twice per day and in another embodiment, the instructions are for daily application only once per day. In one preferred embodiment, the instructions are for treating or preventing a coronary condition.
In one embodiment, the instructions are attached to the container. In another embodiment, the container is a bottle and the dispenser is attached to the bottle. The dispenser can be a pump.
According to another aspect of the invention, another kit is provided. This kit includes a package containing a transdermal patch, and instructions for using the patch to treat a coronary condition. The patch includes a housing, a reservoir in the housing, a membrane attached to the housing, adjacent the reservoir, for placement against the epidermis of a human subject, and an adhesive attached to the housing for holding the membrane to the epidermis of the subject. Progesterone in a carrier is contained in the reservoir, and the patch is constructed and arranged to deliver an amount of progesterone to the epidermis of the subject to achieve blood levels of progesterone of between 0.1 nanograms/ml and less than 4 nanograms/ml for at least 4 hours per day. The amount results in peak blood levels of progesterone of less than 6 nanograms/ml for the period that the subject wears the patch. In a preferred embodiment, the progesterone is delivered in an amount to achieve blood levels of between 1 nanogram/ml and 2 nanograms/ml for at least 6 hours per day, and preferably for between 8 and 24 hours per day. In one important embodiment, the patch delivers between 10 milligrams and 100 milligrams of progesterone to the epidermis over the period, and in another embodiment the patch delivers between 20 milligrams and 50 milligrams of progesterone to the epidermis over the period when applied to the epidermis.
According to another aspect of the invention, a kit is provided for dispensing a pharmaceutical. The kit includes a package housing a plurality of individual dispensing packets, each packet containing a topical preparation of progesterone. The kit also includes instructions for dispensing from one or more of said plurality of packets an amount of the topical preparation which, when applied topically to the skin, achieves blood levels of progesterone of between 0.1 nanogram/ml and less than 4 nanograms/ml for at least 4 hours per day and wherein said amount results in peak blood levels of progesterone of less than 6 nanograms/ml after said application. In important embodiments, each packet contains between 10 milligrams and 100 milligrams of progesterone. In other important embodiments, each packet contains between 20 milligrams and 50 milligrams of progesterone. In certain embodiments, the instructions are for daily application only twice per day, or only once per day.
According to another aspect of the invention, a method is provided for screening the ability of a test compound to inhibit coronary vasospasm. The compound is administered to a coronary hyperreactive animal free of known coronary plaques and coronary injury. Conditions are applied to the animal which, in the absence of an inhibitory compound, would provoke a coronary vasospasm. The presence or absence of coronary vasospasm in the presence of the compound then is determined, the absence being indicative that the test compound is an inhibitor of coronary vasospasm. The preferred animal is a nonhuman primate, preferably a rhesus monkey. The conditions which are applied preferably are a combination of at least two vasoconstrictive agents, and most preferably a combination of serotonin and U46619.
According to still another aspect of the invention, a method is provided for provoking an arterial vasospasm in a non-human animal, preferably a mammal. A vasoconstrictive agent is applied by intracoronary injection (via a coronary catheter) to a coronary hyperreactive animal in amounts effective to produce a vasospasm. In important embodiments, the animal is a nonhuman primate, preferably a rhesus monkey, and the animal can be rendered coronary hyperreactive by ovariectomy. In important embodiments, the arterial vasospasm is a coronary artery vasospasm. Preferably, the vasoconstrictive agent is at least two different vasoconstrictive agents, most preferably serotonin and U46619. Most preferably, the animal is free of known coronary plaques and coronary injury. In one embodiment, the vasoconstrictive agent is applied following or contemporaneously with a vasoprotective agent, such as an estrogen or progesterone.
In another embodiment, the invention is a method for determining the ability of a test compound to inhibit or reduce coronary artery reactivity in vitro. In accordance with this method, vascular muscle cells (VMC) from a coronary artery are isolated. Preferably, the VMC are isolated from coronary hyperreactive monkeys, such as ovariectomized monkeys. The VMC are exposed to an agent known to induce coronary vasospasm, such as a combination of serotonin and a thromboxane A2 mimetic, such as U46619. If the ability of a test compound to prevent vasospasms is to be determined, the VMCs are exposed to the test compound before the induction of vasospasms. If the ability of a test compound to treat, such as to relieve, a vasospasm, the test compound is administered to the VMC following induction of a vasospasm. The VMCs are then observed for the presence or absence of indicia of coronary artery vasospasm. The test compounds may be administered following or at the time of administration of an agent that protects against coronary vasospasm, such as an estrogen or progesterone.
In another embodiment, the invention is a method for producing an in vitro model of a coronary artery vasospasm. A freshly dispersed or primary cultured VMC, preferably from coronary hyperreactive monkeys, is exposed to one or more chemical agents, either alone or in combination. The VMC is then observed for the presence or absence of indicia of coronary artery vasospasm. If desired, the exposure to the test chemical agent may be after or simultaneous with exposure of the VMC to an agent that inhibits coronary vasospasm, such as an estrogen or progesterone.
In any of the foregoing therapeutic embodiments, the progesterone can be applied together with other hormone replacement therapy, particularly estrogen. The progesterone is believed not only to decrease coronary artery reactivity and counter the undesirable effects of estrogens that increase the risk of endometrial hyperplasia and cancer, but it also is believed to assist in retarding the development of osteoporosis and loss of cognitive function in Postmenopausal or ovariectomized women.
These and other aspects of the invention are described in greater detail below.